Regulatory CD4+, CD25+ T cells (T-regs) have risen to the forefront in research investigating self-tolerance. Promotion of transplantation tolerance is a logical application of T-regs, however, this approach is unlikely to succeed until their generation, function, and regulation in vivo are understood. During the initial funding period, we found that T-regs are critical mediators of transplant tolerance induced by intrathymic antigen inoculation. Experiments in the current proposal will define the pathways by which T-regs are generated in vivo, and characterize the mechanism of T-reg induced tolerance. These investigations capitalize on the unique model of T-reg generation we have developed, in which T-regs develop in vivo from an antigen-inoculated thymus. We will test the hypothesis that critical parameters in the selection of T-regs are the APC on which the antigen is presented and the avidity of the interaction with the TCR. In Aim II, we explore the mechanisms by which T-regs inhibit the response of naive T cells to prevent rejection. Preliminary data obtained in the prior funding period indicates that T-regs inhibit the proliferation of naive graft reactive T cells that encounter antigen in the draining lymph nodes. We use this observation as the basis to hypothesize that the interaction between naive and regulatory T cells requires an APC bridge to deliver the suppressive signal and that this interaction results in linked-suppression. Using MHC class II deficient grafts, we examine selectively the impact of T-regs on direct and indirect pathways of alloaggression. Lastly, we explore the fate of regulated T cells, assessing whether these cells undergo programmed cell death or acquire a regulatory phenotype themselves after encountering T-regs. In Aim III, we focus on mechanisms regulating regulatory T cell function in vivo to dissect our provocative observation that acute grafts are more vulnerable than established grafts to the suppressive effects of T-regs. We posit that the inflammatory environment accompanying acute grafting aborts regulatory cell function by activation of graft APC's, specifically dendritic cells. We probe the signals rendering an acutely transplanted graft relatively invulnerable to T-reg mediated suppression and determine the role of Toll receptor mediated activation of graft DC's on T-reg function. In Aim IV, we address practical obstacles that may interfere with the clinical application of T-regs in promoting tolerance to allografts including whether their generation or function is inhibited by commonly used immunosuppressive agents and whether T-regs are equally effective against primed T cells. Collectively, these studies will further our understanding of regulatory T cell function in vivo and help to define their potential for application in the clinical transplant arena.